To discover genes involved in human breast carcinogenesis, we applied DDRT-PCR and identified the candidate human breast cancer-related gene, GIG47 (GenBank accession number AY762102). GIG47 encodes neudesin, a neuron derived neurotrophic factor (NENF) (GenBank accession number NM_013349) in the database.
The neudesin gene has been identified in vertebrates including humans, mice, and zebrafish, but not in invertebrates. This indicates that the neudesin gene is specific to vertebrates. The human neudesin gene is located on chromosome 1 at p33; however, this location has not shown linkage with known inherited diseases [6
]. Neudesin is a novel secreted protein with essentially no primary structural homology to any known proteins. Its activity was governed by the activation of the MAP and PI-3 kinase pathways possibly via
the activation of a Go/Giprotein-coupled receptor. Neudesin is therefore assumed to be a novel neurotrophic factor with a unique structure. Therefore, the study on neudesin may provide new insights into the neuronal development and maintenance [6
However, study demonstrated that GIG47 mRNA is over-expressed in human immortalized cells [10
]. In addition, its protein was also detected as abundant among estrogen receptor (ER)+/progesterone receptor (PR)
breast cancers [11
]. Nevertheless, it is unknown how GIG47 contributes to the cellular and biochemical mechanisms of human tumorigenesis. In this study, our result also revealed that GIG47 is over-expressed in various human tumors including carcinomas of the uterine cervix, lymphoma, colon, lung, skin and leukemia, as well as carcinoma of the breast. In a sharp contrast, expression of GIG47 was generally low in diverse human normal tissues. It implies that GIG47 may play an oncogenic role in multiple body organs. In order to address this, we took advantage of ectopic over-expression system and demonstrated that GIG47, indeed, mediates the tumorigenesis of human breast cancer by promoting the invasiveness of breast cancer cells and tumor growth in vivo
. Therefore, this result strongly supports that GIG47 plays a role in the human tumorigenesis.
Further understanding on GIG47 function and potentially useful information for designing anti-cancer pharmaceuticals may be provided by studying the structural characteristics of GIG47 at an atomic resolution. GIG47 itself shows a poor solubility when it is expressed alone. To obtain a more soluble protein we attached solubility-enhancing tags, which made the total length of the actual protein studied by NMR to consist of 208 residues. Consequently achieving a full resonance assignment was not possible. Nevertheless, as the assigned region of GIG47 has a strong sequence homology of ~50% to a hypothetical protein At2g24940.1 from Arabidopsis thaliana (1TOG) with an unknown function (Figure ), we were able to produce a high-resolution structure of GIG47 by combining NMR chemical shift indices and homology modeling process. Thus our structure determination of GIG47 incidentally made it possible to assign function to a hypothetical protein with an unknown function.
A structure-based rational drug design strategy including SAR-by-NMR screening was successfully applied in developing potent anti-cancer agents based upon inhibitory activity of mdm2 [23
]. Since the potential ligand-binding pocket in GIG47 is hydrophobic in nature like that in mdm2, above strategy may also prove to be useful in developing anti-cancer agents using inhibitors against GIG47. A previous work on NENF (mouse neudesin) showed that the protein has a heme/steroid binding region (Leu22
]. As shown in Figure A, the amino acid sequences of NENF and GIG47 are highly homologous (94.6%). In particular the residues in the heme binding region of NENF are exactly the same as those in GIG47 except for 4 residues. Thus we expect that GIG47 (human neudesin) should be able to form a heme/steroid binding region. Three residues (Y58, Y64, H98) in mouse neudesin play an important role in binding with heme. In order to determine potential heme-binding residues in GIG47 we have used the prediction program, HemeBind [27
], and found that Phe57, Tyr58, Arg60, Tyr64, Ala66, and Leu67 in GIG47 may interact with heme. The structure of GIG47 we have presented in this work reveal that two residues, Tyr58 and Tyr64, are potential heme-binding sites as shown in Figure B, where the side-chains of these two tyrosines are located in the potential heme-binding pocket facing toward the heme. A similar structural pattern is observed in human adrenal inner zone antigen (hIZA), which has a tetrapeptide segment (D99
) that anchors the heme into the hydrophobic pocket [28
]. A similar tetrapeptide segment is found in GIG47, D50
, thus is expected to carry out a similar role of anchoring a heme group into the hydrophobic pocket of GIG47. In GIG47, a third potential heme-binding residue [7
], His98, is located in the opposite side of two Tyr residues and therefore is not likely to contribute to heme binding. The structural analysis described above for GIG47 insinuates that progesterone, a steroid derived from breast tumors, may be the cellular ligand for GIG47.
Figure 6 Comparison of amino acid sequences of GIG47 and NENF and a ribbon diagram of GIG47 showing the potential heme-binding region. (A) Amino acid sequences of GIG47 (human neudesin) and NENF (mouse neudesin) are aligned. The potential heme/steroid binding (more ...)